Methods for increasing genetic progress in a line or breed of swine using sex-selected sperm cells

ABSTRACT

The invention relates to methods of increasing the genetic progress of a line, breed or herd of swine through the use of sex-selected sperm cells in artificial insemination techniques. The invention also encompasses methods of artificially inseminating a swine via deep intrauterine catheter or via a laparoscopic procedure, which allow the use of reduced doses of sex-selected sperm cells.

This application is a continuation of U.S. application Ser. No.15/655,663, filed on Jul. 20, 2017, which itself is a continuation ofU.S. application Ser. No. 14/406,186, filed on Dec. 5, 2014, whichitself is a national stage entry of International Application No.PCT/US2013/044521, filed Jun. 6, 2013, which itself claims priority toU.S. Provisional application Ser. No. 13/840,598 filed Mar. 15, 2013 andU.S. Application No. 61/656,446, filed on Jun. 6, 2012.

FIELD OF THE INVENTION

The invention relates to methods of increasing the genetic progress of aline, breed or herd of swine through the use of sex-selected sperm cellsin artificial insemination techniques. The invention also encompassesmethods of artificially inseminating a swine via deep intrauterinecatheter or via a laparoscopic procedure, which allow the use of reduceddoses of sex-selected sperm cells.

BACKGROUND

There is a need in the swine industry to increase the rate of desirablegenetic change in lines and breeds as well as to lower operational costson breeding and commercial swine farms. The inventions disclosed hereinachieve these goals by allowing the operator of a breeding or commercialfarm to select the sex of offspring swine using sex-selected sperm cellsamples and/or by allowing the use of far fewer genetically superiorboars through the use of reduced sperm cell doses for artificialinsemination procedures via deep intrauterine catheter or laparoscopy.

SUMMARY OF THE INVENTION

One embodiment of the present invention comprises a method of increasingthe genetic progress of a line or breed of swine comprising collecting asemen sample from a boar from said line or breed; sorting said semensample into at least two subpopulations of sperm cells, wherein at least80% of a first subpopulation bears X-chromosomes or Y-chromosomes;inseminating a sow from said line or breed with sperm cells from saidfirst subpopulation; producing one or more offspring from said sow;calculating a selection index for one or more of said offspring; andselecting one or more of said offspring having a higher selection indexcompared to the average selection index for said line or breed to breedwith a swine from said line or breed so as to increase the geneticprogress of said line or breed.

Another embodiment of the invention comprises a method of increasing thegenetic progress of a line or breed of swine comprising collecting asemen sample from a boar from said line or breed; sorting said semensample into at least two subpopulations of sperm cells, wherein at least80% of a first subpopulation bears X-chromosomes or Y-chromosomes;inseminating a sow from said line or breed with sperm cells from saidfirst subpopulation; producing one or more offspring from said sow;obtaining a value for a trait in one or more of said offspring; andselecting one or more of said offspring having a value for said traitthat is greater than or less than the average value for said trait insaid line or breed to breed with a swine from said line or breed so asto increase the genetic progress of said line or breed.

In some embodiments of the invention, semen samples to be used with theinvention are sorted into at least two subpopulations of sperm cells,wherein at least 60%, 60-65%, 65%, 65-70%, 70%, 70-75%, 75%, 75-80%,80%, 80-85%, 85%, 85-90%, 90%, 90-95%, 95%, 95-99% or about 99% of afirst subpopulation bears X-chromosomes and/or Y-chromosomes.

Another method for increasing the genetic progress of a line or breed ofswine comprises preparing one or more embryos or zygotes, either in vivoor in vitro, using a sex-selected sperm cell sample obtained from a boarof said line or breed, and then transferring said one or more embryos orzygotes, by any method known in the art, into a sow for gestation. Incertain aspects of the invention, the egg donor is a sow from a line orbreed. In some specific embodiments of the invention, the embryo orzygote is transferred into or out of a sow using a deep intrauterinecatheter or laparoscopy. This method can also further comprise the stepsof calculating a selection index for one or more of the offspringproduced from said embryo or zygote; selecting one or more of saidoffspring based on said one or more offspring having a higher selectionindex compared to the average selection index for said line or breed;using said one or more offspring having a higher selection indexcompared to the average selection index for said line or breed to breedwith a swine from the same line or breed so as to increase the selectionintensity of that line or breed. Another embodiment of the inventioncomprises using such embryos or zygotes to repopulate a line, breedand/or herd.

Another aspect of the invention comprises selecting an embryo or zygotefor use in the invention based on the presence or absence of a geneticmarker in the embryo or zygote. In certain embodiments, such a geneticmarker can be screened for by removal of one or more blastomeres fromthe embryo or zygote and testing said one or more blastomeres for thepresence or absence of said genetic marker. The genetic marker can be amarker for, or associated with, for example, any of the swine traitsdisclosed herein. In certain embodiments, one or more blastomeres areremoved from the embryo or zygote at the 4-16 cells stage, the 4-10 cellstage or the 4-8 cells stage. Any technique known in the art can be usedfor removal of a blastomere from an embryo or zygote, including but notlimited to those disclosed in U.S. Pat. No. 7,893,315, the disclosure ofwhich is incorporated by reference herein in its entirety. Briefly, oneof the methods described therein comprises immobilizing an embryo andtapping the immobilized embryo until a blastomere is isolated (theembryo can be immobilized using a micropipette and the micropipetteholder is tapped to isolate the blastomere). In certain embodiments,screening said embryo or zygote for a genetic marker comprisesgenotyping a blastomere or cell obtained from said embryo or zygote.Techniques for screening embryos or zygotes for a genetic marker aredisclosed in U.S. Pat. No. 8,338,098, the disclosure of which isincorporated by reference herein in its entirety. Briefly, singlenucleotide polymorphisms (SNPs) can be identified using the pooled DNAsequencing approach, and then genotyping of the identified SNP can beachieved by a PCR-restriction fragment length polymorphism (PCR-RFLP)based method.

The sorted sperm cells used with the invention constitute “sex-selected”sperm cell samples. Sex-selected sperm cell samples can be derived byany technique known in the art. In one embodiment of the invention, thesex-selected sperm cell samples can be prepared using a flow cytometer.The sex-selected sperm cells for use in any embodiment of the inventioncan be cryopreserved using any known method and then thawed before use,or alternatively fresh (i.e., never frozen) sex-selected sperm cells canbe utilized.

The terms “line” and “breed” mean a group of animals having a commonorigin and similar identifying characteristics. The instant invention isalso applicable to “pure lines” and “pure breeds” of swine, as thoseterms are used in the art.

The term “selection index” refers to a numerical score generated for anindividual swine breed based on the swine's expression of certain traitsselected by a breeder. Typically, a breeder will assign a given numberof traits, often referred to as selection objects, to each line or breedof swine, and may further differentially weight the importance eachtrait in generating a selection index. A higher selection index for aswine means that the swine has expressed or carried those traits to agreater degree.

In certain embodiments of the invention, any desirable genotypic orphenotypic trait can be used to construct a selection index for a lineor breed of swine. Phenotypic traits that can be utilized in a selectionindex include, but are not limited to, feed efficiency, average dailygain, carcass lean, carcass quality, fertility, litter size and milkproduction.

In some embodiments of the invention, the selection index for aparticular swine can be calculated using data derived solely from thatparticular swine in, i.e., individual data, or alternatively, theselection index for a particular swine can be calculated using dataderived from a group that contains or is representative of thatparticular swine, i.e. group data. For example, feed efficiency can bemeasured in an individual boar—thus, the selection index for the boar inthis example would be based on individual data. Alternatively, feedefficiency can be measured for the group of boars housed with the boarin question—the selection index for the boar in this example would bebased on group data.

Other aspects of the invention encompass inseminating a sow from saidline or breed with sex-selected sperm cells using a deep intrauterinecatheter or a needle inserted through a membrane of said sow. Some ofthese embodiments encompass known surgical and non-surgical techniquesthat can be used to place sperm cells into a sow's reproductive tract,including laparotomy (surgical procedure involving a large incisionthrough the abdominal wall to gain access into the abdominal cavity).This embodiment contemplates inseminating sows using 1×10⁹ or less totalsperm cells.

In other embodiments, a deep intrauterine catheter can be employed toadminister a sperm cell sample into distal portions of a sow'sreproductive tract such as one or more uterine horns or one or moreutero-tubal junctions. In another aspect of the invention, the deepintrauterine catheter is comprised of an outer tube or sheath and aninner flexible probe. In a further aspect of the invention, the flexibleinner probe comprises a flexible inner duct through which fluids orcells can pass. In certain embodiments of the invention, the outer tubeand inner flexible probe can be made of a plastic, and in otherembodiments, they may be made of metal configured to be flexible such asin a coil or spring configuration. In a further embodiment, the deepintrauterine catheter comprises a video camera or scope for visualizingthe location of the distal portion of the deep intrauterine catheterwithin a sow's reproductive tract. In an alternative embodiment, thedeep intrauterine catheter can be visualized within the sow'sreproductive tract using radiography or fluoroscopy. In anotherembodiment of the invention, a deep intrauterine catheter can be used toinsert or withdraw embryos or zygotes from the distal portions of asow's reproductive tract such as from one or more uterine horns or fromone or more utero-tubal junction.

With respect to insemination with a deep intrauterine catheter, it iscontemplated that a dose of 1×10⁹ sperm cells or less is administered toa sow. Such sperm cells may be sex-selected sperm cells. In oneembodiment of the invention a dose of sex-selected sperm cells (forinstance 600×10⁶, but may be more, or as little as 10×10⁶ if placed inthe optimal location at the optimal time of estrus) is administered intoone or both uterine horns (e.g., 300×10⁶ sperm cells into each horn) ofa sow by deep intrauterine catheter. In other embodiments, doses canvary in the range of or anywhere in between about 300×10⁶, about150×10⁶, about 140×10⁶, about 100×10⁶, about 70×10⁶, about 50×10⁶, orabout 5×10⁶ sex-selected sperm cells or less and can be administeredinto one or both uterine horns of a sow.

The aforementioned doses can be administered in various volumes,including but not limited to 5 ml for every 150×10⁶ sperm cells, or thesame number of cells in a volume in the range of 5 ml, 10 ml, 15 ml, 20ml, 25 ml, 30 ml or 100 ml, or somewhere between 5-10 ml, 10-20 ml,20-30 ml, 30-40 ml, 40-50 ml, 50-60 ml, 60-70 ml, 70-80 ml, 80-90 ml or90-100 ml.

The sex-selected sperm cells for use in any embodiment of the inventioncan be cryopreserved and then thawed, or alternatively fresh (i.e.,never frozen) sex-selected sperm cells can be utilized. Theaforementioned doses may also be administered into one or moreutero-tubal junctions of a sow.

This embodiment of the invention also encompasses the use of alaparoscope to visualize insertion of a needle through a membrane of asow for administering a sex-selected sperm cell sample. Both the needleused for injecting the sperm cell sample and the laparoscope, as well asmanipulating instruments such as forceps, can be inserted into theabdomen of a sow through small incisions typical of laparoscopicprocedures. The invention also encompasses the injection of a sperm cellsample in one or more locations within the female reproductive tract. Byway of example only, the sperm cell sample can be injected in one ormore locations within the uterus of a sow, including one or more uterinehorns, oviducts, ampulla, isthmus or utero-tubal junction. In anotherembodiment of the invention, embryos or zygotes can be inserted orwithdrawn from a sow's reproductive tract via laparoscopy.

With respect to insemination via laparoscopy, it is contemplated that adose of 1×10⁹ sperm cells or less is administered to a sow. Such spermcells may be sex-selected sperm cells. In one embodiment of theinvention a dose of about 500×10⁶ sex-selected sperm cells or less canbe injected into one or both oviducts (e.g., 250×10⁶ sperm cells in eachoviduct) of a sow by laparoscopy; in other embodiments, doses in therange of or anywhere in between about 10×10⁶, about 5×10⁶, about 3×10⁶,about 2.0×10⁶, about 1.2×10⁶, about 1×10⁶, or 0.6×10⁶ sex-selected spermcells or less can be injected into one or both oviducts of a sow.

In a further embodiment, sex-selected sperm cells can be injected intospecific regions of the oviduct, including but not limited to theisthmus, the ampulla and/or the utero-tubal junction. In certainembodiments, a dose in the range of or anywhere in between about 5×10⁶,about 2×10⁶, about 1×10⁶, about 600×10³, about 500×10³, about 300×10³,or about 150×10³ sex-selected sperm cells or less, can be injected intoone or more regions of the oviduct, either unilaterally or bilaterally.

In a further embodiment with respect to insemination via laparoscopy canbe multiply injected at various sites in the oviduct using doses in therange of or anywhere in between about 500×10³ sex-selected sperm cellsinjected into each ampulla with about 1×10⁶ sex-selected sperm cellsinjected into each utero-tubal junction; or a dose of about 1×10⁶sex-selected sperm cells injected into each ampulla with about 2×10⁶sex-selected sperm cells injected into each utero-tubal junction; or adose of about 5×10⁵ sex-selected sperm cells injected into each ampullawith about 2×10⁶ sex-selected sperm cells injected into each utero-tubaljunction; or a dose of about 5×10⁵ sex-selected sperm cells injectedinto each ampulla with about 1×10⁶ sex-selected sperm cells injectedinto each utero-tubal junction. The aforementioned doses can becontained in various volumes, by way of example, 100 μl for every 1×10⁶million sperm cells, or the same number of sperm in one of the followingor in any volume between: 50 μl, 100 μl, 200 μl, 300 μl, 400 μl or 500μl.

Another aspect of the invention comprises synchronizing estrus and/orinducing timed ovulation in a sow that is to be inseminated using theembodiments disclosed herein by administering one or more hormone orhormone analogs to the sow. In one embodiment, the one or more hormoneor hormone analogs comprises PG600 (comprising pregnant mare's serumgonadotropin, “PMSG” and human chorionic gonadotropin, “hCG”; Intervet),OvuGel (triptorelin acetate in a slow release formula via anintravaginal delivery system; Gel Med Sciences, Inc.), equine chorionicgonadotropin, “eCG,” hCG, or progestin.

In a further embodiment of the invention, said one or more hormone orhormone analogs is administered by a programmable device placed in thereproductive tract of said sow. The programmable device contemplatedherein is able to release said one or more hormone or hormone analogs ina time released fashion without the breeder having to monitor the deviceor provide any input other than programming the initial parameters forrelease of said one or more hormone or hormone analog. In anotherembodiment of the invention, estrus synchronization/timed ovulation canbe induced in a sow by administering 1250 to 1500 IU of eCG and then 750IU of hCG 72 to 80 hours later. In another embodiment, estrus can beinduced in a sow by administering 400 to 2000 IU of PMSG and then 500 to1000 IU of hCG is administered 72 to 83 hours later.

Other embodiments further contemplate detecting ovulation in a sow byexamining said sow's follicles. In a particular embodiment of theinvention, said sow's follicles are examined using ultrasound. In afurther embodiment, said sow's ovaries are examined by transrectalultrasound every 3-5 hours beginning 25-35 hours after hCG injection forthe presence of pre-ovulatory follicles. In a further embodiment of theinvention, sows showing multiple pre-ovulatory follicles are selectedfor insemination 2-3 hours after ultrasound.

The invention additionally encompasses a method of increasing the numberof offspring of genetically superior boars in a swine herd or on a swinefarm comprising establishing a subpopulation of one or more geneticallysuperior boars from a population of boars in a herd or on a farm;obtaining sperm cell samples from the one or more genetically superiorboars; preparing a plurality of sperm cell doses from each of the spermcell samples; administering one or more hormone or hormone analogs to aplurality of sows in said herd or on said farm in order to establish aknown time of ovulation for each sow; and inseminating the sows with oneor more sperm cell doses using a deep intrauterine catheter or alaparoscopic procedure, wherein the one or more sperm cell dosesadministered to each sow together comprise a total of less than 1×10⁹sperm cells. These steps can also be used to reduce the number of boarsnecessary for breeding in a swine herd or on a swine farm. In certainembodiments, genetically superior boars comprise boars with a higherselection index relative to other boars within the herd or on the farm.

The invention also encompasses a novel method for increasing theprofitability of a swine herd or farm comprising calculating a netincome per pig for a male pig and for a female pig based on marketconditions to which the herd or farm is subject; determining whether themale pig or the female pig results in a higher net income per pig;collecting a semen sample from a boar; sorting said semen sample into atleast two subpopulations of sperm cells, wherein at least 80% of a firstsubpopulation bears (i) X-chromosomes if the female pig results in ahigher net income per pig, or (ii) Y-chromosomes if the male pig resultsin a higher net income per pig; inseminating a sow with sperm cells fromsaid first subpopulation; and producing offspring from said sow.

As used herein, the term “sow” encompasses gilts (young female pigs thathave not yet farrowed) as well as any reproductively mature female pig.

Any of the embodiments of the invention can utilize a sow that is amember of a genetic nucleus or multiplier herd. Similarly, anyembodiment of the invention can utilize a boar that is a member of agenetic nucleus or multiplier herd.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates schematically a flow cytometer that can be used tosort sperm cell samples into one or more subpopulations bearing X- orY-chromosomes.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention are directed to methods of increasing therate of desirable genetic change or progress in a herd or line of swinethrough the use of sex-selected sperm samples for insemination. The useof sex-selected sperm cells produces offspring that have a highprobability of being either all male or all female, depending on theseparated subpopulation that is used. As shown below, this abilityprovides a significant advantage in the swine breeding industry sincethat industry greatly values annual genetic progress. An additionalbenefit of the use of sex-selected sperm cells is a reduction in theoperational costs associated with maintaining infrastructure andmaterials for both sexes—e.g., the need for separate housing for maleand female swine is eliminated or greatly reduced as well as the need tomaintain two different supply chains for feed for males and females(males are typically fed feed that is higher in protein). A farm whosebusiness is to sell either boars or gilts exclusively can essentiallydouble their productivity using sex-selected sperm cells forinsemination. Alternatively, they can reduce their adult herd inventoryby 50% and hence reduce the working capital investment required.Enabling the use of sex-selected sperm cells in swine also allows abreeder to increase or decrease a trait in a given litter or herd ofswine if that trait is expressed, or expressed more strongly, in one sexversus the other. For example, males are often castrated on commercialfarms in order to reduce “boar taint” of the meat—thus, the ability toproduce only females eliminates the need for castration and completelyeliminates the problem of “boar taint.”

The process of producing sex-selected sperm cell samples, however, istime consuming and expensive, typically requiring the use of specializedflow cytometry equipment, highly trained technicians and complexprocesses. Unfortunately, the typical dose of boar sperm cells requiredfor successful fertilization using conventional artificial inseminationtechniques such as intra-cervical insemination is 1×10⁹ sperm cells to3×10⁹ sperm cells, with the typical boar ejaculate containingapproximately 6×10¹⁰ sperm cells. Therefore, the typical boar ejaculatecontains approximately 20 to 60 artificial insemination doses, greatlylimiting the commercial application of sex-selected sperm cell samplesin breeding swine. Furthermore, females are inseminated a minimum of twotimes per estrus cycle. Accordingly, if the total number of sperm cellsneeded for successful fertilization can be reduced, a greater number ofartificial insemination doses can be produced for a given boar in agiven amount of time, making the use of sex-selected sperm cell samplesmuch more desirable from a commercial standpoint. In order to optimizethe commercial application of sex-selected sperm cell samples in swine,the instant invention also encompasses methods for reducing the totalnumber of sperm cells in a sample needed for an efficacious artificialinsemination dose, including insemination via deep intra-uterinecatheter and laparoscopy. A reduced number of sperm cells required forsuccessful fertilization produces the additional benefit of reducing thenumber of boars, and their associated costs, needed for breeding.

A. Increasing the Rate of Genetic Change

By using the instant invention to select the sex of the offspring in aline, the rate of desirable genetic change of a herd or line can beincreased. Genetic change, or genetic progress, in this context meansthat for a given trait, the individuals in the successive generationwill express the desired trait more strongly than the previousgeneration. With respect to undesirable traits, genetic progress meansthe individuals in the successive generation will express the trait lessstrongly than the previous generation.

Genetic progress from one generation to the next (ΔG) can be measured asthe difference between the average genetic level of selected parents andthe average genetic level of the selection candidates (the animalsavailable for selection). In ideal conditions, this depends upon theheritability (h²) of the trait and the difference between the averageperformance of selected parents (P) and that of selection candidates(A). The difference between the average performance of selected parents(P) and that of all selection candidates (A) (of which the selectedparents are a subset) is also known as the selection differential (SD).ΔG=h ²(P−A)

The sign in front of the selection differential indicates the directionof selection—a positive value indicates selection for larger phenotypicvalues while a negative selection differential would indicate selectionfor smaller phenotypic values. Thus, if the goal is selection for largerphenotypic values, individuals with large positive selectiondifferentials relative to other individuals within the line, breedand/or herd are deemed to be superior individuals and can be selected asparents on that basis. Similarly, if the goal is selection for smallerphenotypic values, individuals with large negative selectiondifferentials relative to other individuals within the line, breedand/or herd are deemed to be superior individuals and can be selected asparents on that basis.

Selection is more effective when non-genetic effects are removed (e.g.by comparing each performance record to the average of the contemporarygroup) and when information from relatives is used in addition to thatof the animal itself. This is achieved through the computation ofestimated breeding values (EBVs). When selection is based on EBVs, theexpected genetic progress is equal to the difference between the averageEBV of the selected animals and that of the selection candidates.ΔG=P _(EBV) −A _(EBV)

The annual rate of genetic progress is then: ΔG/t=(P_(EBV)−A_(EBV))/t,where t is the generation interval. In other words, the annual rate ofgenetic progress depends on the generation interval and on thesuperiority of the parent's EBVs compared to that of the selectioncandidates. In certain embodiments of the invention, a statistical modelsuch as best linear unbiased prediction (BLUP) can be used to determineEBVs, although any statistical model known in the art can be implementedfor use with the invention.

Typically, when breeding swine, more than one trait is selected for.When there are multiple traits to be selected for, however, selectionmust be balanced for each trait depending on their economic values, howwell they respond to selection (heritability) and how they influenceeach other (genetic correlations). One way to achieve such a balance isto create a selection index, which depends on the above values and theEBVs for each trait for each animal. The prediction of genetic progressfor a selection index is the same as for an individual EBV, i.e., theannual rate of change in the index is a function of the generationinterval and of the difference between the index of selected parents andthe index of selection candidates.

In a large population, the selection differential depends upon how manyanimals are tested and how many are selected—the lower the proportionselected the higher the selection differential. Thus, in order tomaximize genetic progress, one should rank all tested animals based onthe selection index and then select the minimum number of top boars andsows required to maintain the line, breed and/or herd size. This ensuresthat the average index of selected animals is substantially higher thanthe average index of all animals tested.

As shown above, genetic progress is dependent on identifying superiorindividuals as parents for the next generation. There are two methodsused to measure the phenotypic superiority of a selected individual:selection differential, as discussed above, and selection intensity. Thetwo measures are closely related for traits with values distributed asexpected for the normal distribution. The selection differential isequal to the product of the selection intensity and the phenotypicstandard deviation. Because selection intensity is in standard deviationunits, it is possible to compare selection pressures for differenttraits regardless of the type of units used to measure performance.

Additionally, one can convert the proportion of candidates selected asparents into selection intensity, since there is a direct relationshipbetween proportion of a population and number of standard deviationsbetween means in a normal distribution. Thus, the smaller the proportionof the population selected, the larger the selection intensity and thelarger the genetic progress, all else being equal.

Furthermore, in order to achieve a greater rate of genetic progress, onewould also want to keep the generation interval small—generationinterval is the average age of parents in the herd when their progenyare born and can be decreased through faster replacement of boars andsows in the line, breed and/or herd. To keep the generation interval lowin swine, boars should be culled before they are one year of age andsows after one or two farrowings. Finally, in order to increase geneticprogress, the selected trait or traits should have a high level ofheritability. The heritability of a trait is the proportion ofobservable differences in a trait between individuals within apopulation that is due to genetic, as opposed to environmental,differences.

Examples of important traits in the swine industry are feed efficiency,i.e., a measure of an animal's efficiency in converting feed mass intoincreased body mass (also known as feed conversion or feed to gainratio), and average daily gain, i.e., the average daily weight gain foran animal. Traits are measured in different units (e.g., number of pigs,pounds per day, inches, etc.), are not of equal economic importance inall global markets and are not genetically influenced to the same degree(i.e., different heritabilities). Generally speaking, production traitssuch as feed efficiency and average daily gain have high heritability.In contrast, reproductive traits such as fertility and litter sizegenerally have low heritability.

In certain embodiments of the invention, the efficiency with which aboar's sperm cells can be separated into X-bearing and Y-bearingsubpopulations is a trait that can be selected for in a line, breed orherd. Increased expression of this trait allows the X-bearing andY-bearing subpopulations to be sorted at an increased rate and/or withhigher purity levels as compared to the average for individuals in theline, breed and/or herd, all else being equal. Certain features of aboar's semen sample can be measured to assess the efficiency with whichits semen can be sorted, including the number of dead sperm in theunprocessed ejaculate, as well as how well the sperm cells take up aDNA-selective dye, such as Hoechst 33342, to create saturation of thestaining process used when sorting with a flow cytometer. The number orpercentage of dead sperm cells in a semen sample can be assessed using acolored or fluorescent dye that preferentially binds to damaged or deadcells, for example. Superior dye saturation is associated with asuperior split of the X- and Y-chromosome bearing subpopulations asobserved on the flow cytometer and/or a superior “peak to valley” ratiofor the histograms generated for the X- and Y-chromosome bearingsubpopulations by the flow cytometer.

Swine production can be represented by a multilevel pyramid, withcertain offspring at each level used in the next lower level forbreeding. The top level of the pyramid is the nucleus herd. The nextlevels from top to bottom are the daughter nucleus herd, the multiplierherd and finally the commercial farm, respectively.

A nucleus herd is typically comprised of 12 to 15 lines, although anynumber of lines may be represented, with each line comprising a numberof desirable traits.

Each line can also be assigned its own selection index based on traitsselected by a breeder for that line, against which offspring within eachline are assessed—offspring having a higher selection index score beingmore desirable. As noted above, the selection index for a particularswine can be calculated using individual data or group data. The purposeof a selection index is to assign appropriate emphasis to each of thevarious traits to provide a single value for use in comparing differentanimals. Only the offspring with the highest measured selection indexare retained in the nucleus herd. Sows within each line are eitherboar-line mothers, i.e., used to produce males, or gilt-line mothers,i.e., used to produce females. Female offspring of boar-line mothers arediscarded. Likewise, male offspring of gilt-line mothers are discarded.

Accordingly, using sex-selected sperm cells of the invention forinsemination, the offspring of boar-line sows have a greater chance ofbeing male and the offspring of gilt-line sows have a greater chance ofbeing female as compared to using conventional, unsorted semen, therebysignificantly raising the selection intensity of the respective linessince there is a larger population of offspring of the correct sex tomeasure and then select parents from. For example, a sperm cell sampletaken from a boar-line boar can be separated using the techniquesdisclosed herein to create a subpopulation in which at least 80% of thesperm cells bear Y-chromosomes (when using the invention on a gilt-line,a subpopulation in which at least 80% of the sperm cells bearX-chromosomes can be used). That subpopulation, or a portion of thatsubpopulation, can then be used to inseminate a boar-line sow. Becausethere is at least an 80% chance the offspring from that sow will bemales, the selection intensity of the boar-line will be increased sincethere will be more males to select from.

At the daughter nucleus level or “line multiplication” level, sows froma line are inseminated with boar semen derived from the nucleus herd. Atthe multiplier herd level, the resulting sows are typically crossed withboars of a different line. A multiplier herd is typically either a giltmultiplier or boar multiplier. Gilt multipliers produce parent gilts andboar multipliers produce parent boars. Parent gilts are sent tocommercial farms to replace old or dead gilts and sows, and parent boarsare sent to boar studs to produce the sperm cells for use in artificialinsemination.

B. Changing the Expression of Traits Across a Litter or Herd

As noted above, the use of sex-selected semen in swine breeding allowsone to increase (or decrease) the expression of certain traits in agiven litter or herd if those traits are expressed, or expressed morestrongly, in one sex versus the other.

For example, gilts have higher feed conversion than barrows (male pigsthat have been castrated before sexual maturity). Since feed costsrepresent a significant proportion of the cost of swine production,having litters comprised of all or substantially all females may resultin increased profitability for the breeder or farm depending on localmarket conditions. Furthermore, compared to barrows (young castratedmales), gilts (young females) generally have lower body weight, loweraverage daily gain, lower average daily feed intake and are leaner (lessbackfat depth, increased loin depth, higher fat-free lean index). Thus,although gilts may grow more slowly than barrows, they will be leanerand more efficient and thus may be more profitable for a farm under theright market conditions.

Accordingly, the use of sex-selected semen in swine can allow one toincrease the profitability of a farm by selecting the sex having themost desirable traits under the market conditions.

Example 1—Preparation of Sex-Selected Boar Sperm Cell Samples

The following process for the preparation of a sex-selected boar spermcell sample is provided by non-limiting example only. The first step inthe manufacture of sex-selected boar sperm cell sample is to obtain anejaculate from a suitable boar. Once the ejaculate has been collected,it can be extended in a suitable extender, that may include anantioxidant. A sperm rich fraction of the ejaculate can then be diluted.If the sample needs to be transported prior to sex-selection, the samplecan be held at a temperature of 0-39° C. (typically 16-17° C.) forbetween about 12 hours to about 18 hours while it is being shipped fromthe collection point to the flow cytometer for the sex-sorting process.

Once the sperm cell sample is in the laboratory, various quality checkscan be conducted on the sperm cell sample including checking themotility (e.g., via CASA System), viability (e.g., via flow cytometer),morphology (e.g., via microscopy) and concentration (e.g., viaNucleoCounter). Sperm cell samples that pass these quality checks arethen prepared for sorting.

Prior to putting the sample through the flow cytometer, the sample isstained with a DNA selective dye, exposed to a quenching dye to form astained sperm cell sample, which is subsequently placed into a spermcell source of the flow cytometer. Specifically, the sperm cell samplein some embodiments, can be first diluted with a buffer or extender,such as BTS (see Table 1) to a final concentration which in some casescan be 100×10⁶ cells/ml, and the DNA selective dye Hoechst 33342 (can be5 mg/ml in MiliQ water; Ref: B-2261) is then added, a good workingconcentration can be about 5 μl/100 million cells/ml but DNA dye can beused at lower and higher concentrations in the range of 0.5 to 20 ul/100million cells/ml. The sample is then usually placed in covered bathwater between 30 and 42° C. (usually close to 35° C.) for between 10 minand 12 hours, with exceptional staining at about 50 minutes, and thensubsequently placed in a dark area at room temperature (21-22° C.) priorto sorting. Before sorting the sample, the sample is filtered to removelarge debris and cells (for example with CellTricks of 0.30 μm) andafter filtering, red food dye may be added (when added, usually 0.5-5μl, or 1 μl of a 25 mg/ml stock solution in MiliQ water) or anotherquenching dye, can be added to the sample. The sample can then be sortedusing a flow cytometer with a sheath fluid which in some cases maycomprise the components as listed in Table 2, but other sheath fluidsmay be used as well.

FIG. 1 illustrates, in schematic form, part of a flow cytometer used tosort a sperm cell sample to form one or more subpopulations, the flowcytometer being generally referenced as 10. In this particularembodiment, sex sorting is taking place, so the subpopulations areX-chromosome bearing sperm cells and Y-chromosome bearing sperm cells.FIG. 1 represents a single technique for sorting sperm, but any knowntechnique for sorting cells known in the art can be used with certainembodiments of the invention.

The flow cytometer 10 of FIG. 1 can be programmed by an operator togenerate two charged droplet streams, one containing X-chromosomebearing sperm cells, charged positively, 12, one containing Y-chromosomebearing sperm cells, charged negatively 13 while an unchargedundeflected stream of dead cells 14 simply goes to waste.

An operator may also choose to program the flow cytometer in such amanner, that both the X- and Y-chromosome bearing sperm are collectedusing a “high purity sort” (in other words only live X- and Y-chromosomebearing sperm are collected) or to program the flow cytometer to collectboth the X- and Y-chromosome bearing sperm using an “enriched sort” (inother words it will collect droplets containing live that were notpreviously sorted and excluding all initial dead again by the use ofBoolean Gate logic available with the computer that controls the flowcytometer). The Boolean Gate logic can also be used to collect only oneof either the X- or Y-chromosome bearing sperm.

Initially, a stream of sperm cells under pressure, is deposited into thenozzle 15 from the sperm cell source 11 in a manner such that they areable to be coaxially surrounded by a sheath fluid supplied to the nozzle15 under pressure from a sheath fluid source 16. An oscillator 17 whichmay be present can be very precisely controlled via an oscillatorcontrol mechanism 18, creating pressure waves within the nozzle 15 whichare transmitted to the coaxially surrounded sperm cell stream as itleaves the nozzle orifice 19. As a result, the exiting coaxiallysurrounded sperm cell stream 20 could eventually and regularly formdroplets 21.

The charging of the respective droplet streams is made possible by thecell sensing system 22 which includes a laser 23 which illuminates thenozzle exiting stream 20, and the light emission of the fluorescingstream is detected by a sensor 24. The information received by thesensor 24 is fed to a sorter discrimination system 25 which very rapidlymakes the decision as to whether to charge a forming droplet and if sowhich charge to provide the forming drop and then charges the droplet 21accordingly.

A characteristic of X-chromosome bearing sperm is that they absorb morefluorochrome dye than Y-chromosome bearing sperm because of the presenceof more DNA, and as such, the amount of light emitted by the laserexcited absorbed dye in the X-chromosome bearing sperm differs from thatof the Y-chromosome bearing sperm and this difference communicates tothe sorter discrimination system 25 the type of charge to apply to theindividual droplets which theoretically contain only a single X- orY-chromosome bearing sperm cell. Dead cells (or those about to die)typically absorb the quenching dye which is communicated to the sorterdiscrimination system 25 not to apply a charge to the dropletscontaining such cells.

The charged or uncharged droplet streams then pass between a pair ofelectrostatically charged plates 26, which cause them to be deflectedeither one way or the other or not at all depending on their charge intorespective collection vessels 28 and 29 to form respectively a genderenriched population of X-chromosome bearing and a gender enrichedY-chromosome bearing sperm cells having a DNA selective dye associatedwith their DNA. The uncharged non-deflected sub-population streamcontaining dead cells (or those about to die) go to the waste container30.

The sex-selected sperm cell sample is collected in a 50 ml tube with 2.5ml of catch fluid, which in some embodiments can be TesTrisGlucose (TTG)(see Table 3) with 2% of egg yolk, for every 20 million cells. In thisembodiment, the sex-selected sperm cell sample will typically have afinal volume of approximately 24 ml at about 1×10⁶ cells per ml. Thistube is then stored at room temperature in a dark room for about 2hours.

TABLE 1 BTS Extender CHEMICALS SYGMA CODE g/liter Glucose G6152 36.941Sodium Citrate S4641 5.999 Sodium Bicarbonate S5761 1.261 EDTA ED2SS1.250 Potassium Chloride P3911 0.7456 Kanamycin sulfate K4000 0.05

TABLE 2 Sheath Fluid (PBS) CHEMICALS SYGMA CODE g/liter Sodium ChlorideS9888 8 Potassium Chloride P3911 0.2 Sodium phosphate monobasicmonohydrate S9638 0.12 Sodium phosphate dibasic heptahydrate S9390 1.717EDTA acid E6758 1 Penicillin G potassium salt PENK 0.058 StreptomycinSulfate S6501 0.05

TABLE 3 TesTrisGlucose (TTG) CHEMICALS SYGMA CODE g/100 ml TES T1375 5TRIS T1503 0.68 GLUCOSE G6152 0.6 KANAMICYN K4000 0.005

Once the sex-selected sperm cell sample has been obtained, it can beused with conventional artificial insemination procedures, such asintra-cervical insemination, in vitro fertilization or artificialinsemination with deep intrauterine catheter or laparoscopy.Alternatively, the sex-selected sperm cell sample can be cryopreservedfor storage and then subsequently thawed out for use at a later time.

Example 2—Cryopreservation of Sex-Selected Boar Sperm Cell Samples

Once the sex-selected boar sperm cell sample has been manufactured, thesperm cell sample can be optionally cryopreserved for transport orstorage for use at a later time. The following method of freezing can beused with the invention but is presented by way of example only—anycryopreservation method known in the art can be used.

After sorting, the 50 ml tubes containing the sex-selected sperm cells(with 20 million cells) can be divided into tubes of 15 ml, withapproximately 12 ml of a sex-select sperm cell sample semen in eachtube, each containing approximately 10 million sex-selected sperm cells.Theses tubes can be centrifuged at 3076 g at 21° C. for 4 minutes. Thesupernatant decanted, and the pellet can remain with some of thesupernatant in approximately 50 μl.

To each pellet, a first freezing medium, that may comprise a solution of20% egg-yolk and 80% β-Lactose, can then be added at room temperature.The motility of the sperm cells can then be checked. If acceptable, thetubes can be taken to a programmable temperature control machine(PolyScience—MiniTube) or can be manually handled to decrease thetemperature from about 21° C. to about 5° C. over a period of about 2hours. After the timed temperature shift, the samples can be placed in acold room at about 5° C. where a second freezing medium, which maycomprise egg-yolk, β-Lactose, Glycerol and Equex Stem, or may justcomprise a cryopreservative such as glycerol, or the cryopreservativewith an osmotic stabilizer which is previously cooled to 5° C. is addedto the samples. After 10 minutes, the sex-selected sperm cell samplescan be placed in artificial insemination straws, and the straws thenexposed to liquid nitrogen vapors (approximately 4 cm from the liquidnitrogen) for a short period of time (e.g. 10 minutes) and then placeddirectly into the liquid nitrogen for long term preservation.

When the sex-selected semen samples are ready for use, the straws can beunfrozen by thawing/warming the straws (e.g. place in a water bath setat about 37° C. for about 15 seconds). Post-thaw, motility and viabilityof the sperm cells can then be analyzed at 30, 90 and/or 150 minutes forstandard comparisons.

Example 3—Estrus Synchronization

The invention contemplates that for convenience purposes, estrus can besynchronized and/or timed ovulation induced in one or more sows to beinseminated. Furthermore, because sex-selected sperm is oftenpre-capacitated, it is important to inseminate a sow withinapproximately 6 hours of ovulation. Synchronized estrus or timedovulation helps assure this will be the case. Generally speaking thisentails administering one or more hormone or hormone analogs to thesow(s) to be inseminated. There are several ways to induce estrus/timedovulation in gilts, which are described below.

The one or more hormone or hormone analogs can be administered to thesow in order to establish estrus synchronization as well as time ofovulation. These hormones and hormone analogs typically include, forexample, PG600, OvuGel, eCG, hCG, and/or progestin, and can beadministered manually with timed injections or with the assistance of aprogrammable device placed in the reproductive tract of the sow. Theprogrammable device contemplated herein releases one or more hormone orhormone analogs in a time released fashion without the breeder having tomonitor the device or provide any input other than programming theinitial parameters for release of said one or more hormone or hormoneanalogs. Any of the following methods for inducing and/or synchronizingestrus known in the art may be used generally with the invention,including the following.

(a) Transport and Boar Induced Estrus.

Gilts typically attain puberty at approximately 180-210 days of age.However, the natural attainment of puberty is influenced by manyintrinsic and extrinsic factors, such as genotype, environment and boarcontact. Many breeders and farmers indicate that the first estrus iscommonly observed when gilts are six months of age. The onset of estrusoften coincides with relocation or transport of animals from the giltmultiplier to the commercial farm. Undoubtedly, the best-known stressfactor in pigs is that of transportation. If the age of gilts at thetime of transport is close to the normal onset of puberty, approximately25-35% of gilts will display estrus within one week after transport.This transport-induced estrus can serve to synchronize a proportion ofgilts.

Although transport may induce estrus, it is evident that boar contact isa potent form of puberty stimulation. The major factor controlling theefficiency of boar contact as a puberty stimulus is the age of the giltat the time of boar introduction. When boar contact is initiated whengilts are 4 months of age, the pubertal response is minimal. It wassuggested that the young gilt may become habituated to the boar stimulusat a stage in development when she is too young to respond. Conversely,when boar introduction is delayed until the immediate prepubertal period(6 months of age and above), the response is again limited for adifferent reason. By virtue of the relatively old ages, i.e. 6 months,of gilts at introduction, the actual pubertal ages of these gilts arenot much reduced below those of unstimulated animals. When boarintroduction occurs at gilt ages in the region of 160 days, both theinterval from first boar contact to puberty and gilt age at puberty areminimized, while maximum synchronization of the pubertal estrus occurs.

(b) Oral and Time-Release Progestins.

This approach to estrus synchronization utilizes suppression of ovarianactivity through the administration of orally administered progesteroneor synthetic progestins. Some progestins can be obtained that aretimed-release injectable forms, such as altrenogest (see below). Feedingcyclic gilts individually or in groups at a rate of 15-30 mgaltrenogest/pig/day for 14 to 18 consecutive days produces a synchronousonset of estrus between 2 and 8 days after the last progestin feeding.

(c) Gonadotropins.

eCG/hCG (PG600R) Presently, the most common exogenous hormonecombination for induction of follicle growth and ovulation in acyclicfemales is a combination of eCG, formerly called pregnant mare's serumgonadotropin (PMSG), and human chorionic gonadotropin (hCG). The productPG600R contains 400 IU PMSG and 200 IU hCG. This hormone can bepurchased as a combination drug and is cost-effective for the inductionof estrus and ovulation in acyclic pigs. Gilts usually show estrus 3-6days after treatment and the time of ovulation is approximately 110-120hours. The response rate is enhanced if gilts are given daily boarcontact, beginning at the time of treatment. PG600 comprises pregnantmare's serum gonadotropin, otherwise known as equine chorionicgonadotropin (“PMSG” or “eCG”) and human chorionic gonadotropin (“hCG”)(Intervet). OvuGel is another commercially available gonadotropin(triptorelin acetate) in a slow release formula which can beadministered via an intravaginal delivery system (Gel Med Sciences,Inc.).

(d) Prostaglandins.

PGF₂ alpha is effective for inducing luteolysis, abortion, and a promptreturn to estrus in pregnant (and pseudopregnant) gilts beyond thesecond week of pregnancy. One method for synchronization is to pen-mategilts for three weeks and then, treat with PGF₂ alpha two weeks later.

(e) Time-Release Hormones.

Another method involves the direct injection of a commercially availablepreparation, such as altrenogest or regumate, at a specific time pointin the estrus cycle. For example, in one embodiment of the invention,synchronization and timed ovulation is achieved by administering on day11-14 of a gilt's estrus cycle, 15-30 mg altrenogest/day for 4 to 7days. 24 hours after stopping altrenogest, 400 to 2000 IU of PMSG can beadministered, and then 500 to 1000 IU of hCG, 72 to 83 hours later.

Example 4—Ovulation Detection

Ovulation detection in a sow can be done by examining the sow'sfollicles. The realization of the importance of establishing an adequatesperm reservoir in the oviduct at an appropriate time relative toovulation is critical in the management of artificial insemination inswine. In particular, knowledge of when a sow is likely to ovulateduring estrus is highly beneficial to achieving successful insemination.To that end, in a particular embodiment of the invention, sow'sfollicles are examined using ultrasound after the induction of estrus.In a specific embodiment of the invention, the sow's ovaries areexamined by transrectal ultrasound every 4 hours beginning 30 hoursafter hCG injection for the presence of pre-ovulatory follicles. Sowsshowing multiple pre-ovulatory follicles (diameter of antrum >6 mm) areselected for insemination 2-3 hours after ultrasound.

Example 5—Insemination Using Laparoscopy or Deep Intrauterine Catheter

Once the sex-selected boar semen sample has been prepared, the samplecan be used to inseminate a sow. Any conventional artificialinsemination technique can be used in the invention, includingintra-cervical insemination. However, deep intrauterine catheters andlaparoscopy are particularly relevant in swine, since they allow for theuse of a reduced dose of sperm cells for successful fertilization, inpart because they are able to place the sperm cells in key areas of thesow's reproductive tract, including but not limited to the uterinehorns, the oviducts, the ampulla, the isthmus and the utero-tubaljunction. The use of reduced sperm cell doses allows the use of farfewer genetically superior boars for breeding purposes, which has thebenefits of reducing costs to breeders and reducing the environmentalharm that results from having to maintain a large number of boars.

(a) Insemination Using Deep Intrauterine Catheter. The use of a deepintrauterine catheter allows one to place sperm cells into the uterinehorns of the sow and ideally at the utero-tubal junction. The use andconstruction of such a deep intrauterine catheter is disclosed in U.S.Pat. No. 6,695,767, the disclosure of which is hereby incorporated byreference in its entirety. Such a deep intrauterine catheter canoptionally comprise a video camera or scope to allow the operator to seethe path of the catheter, so that a choice between placing sperm cellsin one or both of the uterine horns can be made. Alternatively, thelocation of the deep intrauterine catheter can be visualized within areproductive tract of a sow when used in conjunction with a radiographicor fluoroscopic device. Because of its length, a deep intrauterinecatheter allows the operator to reach distal regions of a sow'sreproductive tract, including the uterine horns—regions that would beunreachable using a standard catheter used for artificial insemination.In one embodiment of the invention, the length of the deep intrauterinecatheter is 1.8 m, 1-2 m, 1-2.5 m or 1-3 m.

The deep intrauterine catheter can be introduced inside of the cervicalduct of a sow in estrus which may be superovulated but may also benaturally cycling or otherwise induced. A non-toxic lubricant liquid canbe applied onto the catheter to facilitate its passage through thevagina. The catheter can comprise an outer tube or sheath and a flexibleprobe within the outer tube or sheath. In one embodiment of theinvention, once the catheter has been advanced to the cervical duct, theflexible probe can be further advanced within the outer tube of thecatheter. The flexible probe can be advanced until reaching the anteriorportion of a uterine horn. When the flexible probe is advanced withinthe uterine horn, it can bend and thus continue to follow the tortuouspath of the uterine horn. Although it is not absolutely necessary,introduction of small volumes of liquid through the outer tube of thecatheter can facilitate progression of the flexible probe at its passagethrough the cervical duct and its progression through the uterine horn.Once the flexible probe has been introduced up to its final positionwithin the uterine horn, a sperm cell sample contained in a syringebeing connected to the proximal end of the flexible probe and can beintroduced—through a flexible duct within the flexible probe—into theuterine environment. So as to avoid losses of sperm cells and to ensurethat the sperm cell sample has been completely evacuated from theflexible duct, a small volume of liquid can be subsequently introducedthrough the flexible duct. Thereafter, the catheter, comprising theouter tube and the flexible probe, can be withdrawn. In another aspectof the invention, this process can also be used for transferring embryosinto a uterine horn or removing embryos from a uterine horn.

(b) Insemination Using Laparoscopy. Use of laparoscopy to inseminate asow has the advantage that the placement of sperm cells within the sow'sreproductive tract can be even more precise than with the use of acatheter, thus further enabling the use of reduced sperm cell doses forinsemination. Specific areas of the uterus can be targeted, such as theoviduct, the isthmus, ampulla, or the utero-tubal junction. By way of anon-limiting example, the following procedure can be used with theinvention to inseminate a sow via laparoscopy.

For example, a 50 ml tube containing 24 ml of a sex-selected sperm cellsample having about 1×10⁶ sperm cells per ml can be divided into 2 tubesof 15 ml and centrifuged at about 3076 g at a temperature in the rangeof about 21° C. for several minutes (2-5 or 4 minutes). The supernatantcan be recentrifuged under the same conditions if needed. The resultingsemen pellets are then mixed and the concentration checked (viaNucleoCounter). The concentrated sex-selected sperm cell sample is thendiluted with BTS to a final concentration of 10×10⁶ cells/ml and themotility and viability of the sperm cells is checked. (The sperm cellsample should be maintained at room temperature (21° C.) during theentire process.)

Sows can be grouped or separated, for instance they can be allocatedindividually to stalls in a mechanically ventilated confinementfacility. Sows (2-6 parity) are weaned at about 21 days. Estrus can thenbe induced by injecting each female intramuscularly with about 1250 IUequine chorionic gonadotrophin (eCG; Folligon, Intervet InternationalB.V., Boxmeer, The Netherlands—or an equivalent compound) 24 hours afterweaning; 72 hours later, they are treated with about 750 IU humanchorionic gonadotrophin (hCG; Veterin Corion, Divasa, Farmavic S.A.,Barcelona, Spain) or an equivalent. Estrus detection is performed once aday (for instance at 7:00 a.m.), beginning 2 days after eCG injection.One way to detect estrus is to allow females nose to nose contact with amature boar and by applying back pressure, to identify sows that exhibita standing heat reflex, which are considered to be in estrus; at whichpoint the ovaries can be scanned. The ovaries can be examined atperiodic intervals (e.g. every 4 hours) for mature follicles starting atabout 30 hours after hCG injection by transrectal ultrasonography usinga 5 MHz multiple scan angle transducer, to look for the presence ofpre-ovulatory follicles. Only sows showing multiple pre-ovulatoryfollicles (diameter of antrum >6 mm) are selected for insemination.Inseminations are carried out within 2-3 h after ultrasonography.

Laparoscopic inseminations can then be performed on these sows oncesedated (which may be by azaperone administration; Stresnil; 2 mg/kgbody weight, i.m.). General anesthesia can also be induced with acompound such as sodium thiopental (Abbot; 7 mg/kg body weight, i.v.)and maintained with halothane (3.5-5%) or a similar compound. Forsurgery, the sow can be placed in the supine position, and if available,on her back in a laparoscopy cradle. If a cradle is used, it is placedin a Trendelenburg position (hind quarters upward, with the headpointing down) at an angle of approximately 20° above horizontal.

In one embodiment, an incision (about 1.5 cm) is made close to theumbilicus. The edges of the incision can then be pulled up withcountertraction and a 12 mm Optiview trocar (Ethicon Endo-surgeryCincinnati Ohio, USA) with an inserted 0° laparoscope is advanced intothe wound. At the umbilicus, the subcutaneous fatty tissue, the anteriorfascia of the rectus muscles, the rectus muscles, the posterior fasciaof the rectus muscles, the transversalis fascia and the peritoneum aretraversed by slight cutting and moderate pressure. The process iscontrolled via monitor feedback. Although the CO₂ tubing is connected tothe trocar, inflation does not begin until the peritoneum is punctured.After the peritoneal cavity is entered and the pneumoperitoneum started,the handpiece of the Optiview is removed and replaced by the 0°laparoscope. The abdominal cavity is inflated to 14 mmHg with CO₂. Twoaccessory ports are placed in the right and left part of the hemiabdomen, which provides access for laparoscopic Duval forceps formanipulating the uterine horn and grasping the oviduct for theinsemination needle, respectively. The oviduct is grasped with the Duvalforceps in the isthmus region. Then the dose-flow (containing 0.3-0.5million of spermatozoa in 0.1 ml) is inserted, and sex sortedspermatozoa are flushed into the oviduct. The procedure is then repeatedon the other oviduct. After both oviducts are inseminated, the trocarsare removed, and incision wounds sutured.

Example 6—Selection Indexes

Environmental differences make it difficult to compare pigs tested atdifferent locations, at different times, or under different management.The use of selection indexes based on contemporary group comparisons,however, removes much of the influence of these environmental factors.Thus, more valid comparisons of genetic merit are possible. Anyselection index known in the art can be used as a component of theinvention. The following selection indexes are provided by way ofexample only.

Sow Productivity Index (SPI). This index provides a measure of sowproductivity and is useful when culling sows. Prolificacy is measured bythe adjusted number of pigs born alive in a litter. Milking ability ismeasured by the adjusted weight of the litter at 21 days of age.

Early Weaning Sow Productivity Index (EWSPI). This index is designed foruse in culling sows when 21-day litter weights are not available. Litterweight at 21 days is used as a correlated trait when the index isconstructed, allowing some selection emphasis to be placed on milkingability even when weights are not collected.

Maternal Index (MI). The maternal index is intended to put emphasis onmaternal characteristics and is useful for selecting boars to producereplacement gilts and in selecting replacement gilts. Because barrows,and gilts that are unacceptable for replacements, are residuals of thistype of mating, there is some emphasis on growth rate, backfat and feedefficiency. Feed efficiency is included as a correlated trait, althoughit is not measured directly. In certain embodiments, it is recommendedthat potential replacements not be weaned before 10 days of age so thatlitter weight can be used to select for milking ability.

Terminal Indexes (TI). The terminal indexes put emphasis on growth,efficiency, and backfat. The terminal indexes can be used for selectinganimals to be used in terminal crosses. If backfat is measured usingA-mode ultrasound, the TI-A should be used. If backfat is measured withB-mode ultrasound or metal probe, the TI-B is the appropriate index. TheTI-M is for use if predicted percent lean has been calculated.

The above indexes will average 100 for each test group and should have astandard deviation of about 25.

The traits used in the calculation of the above selection indexesdisclosed herein are defined as follows:

L=the adjusted number born alive record on the dam minus the average ofthe adjusted number born alive records of her contemporary group;

W=the adjusted 21-day litter weight record on the dam minus the averageof the adjusted 21-day litter weight records of her contemporary group;

D=adjusted days to 250 pounds measured on the individual minus theaverage of the adjusted days to 250 pounds of the test group;

B=backfat measured on the individual, adjusted to 250 pounds, minus theaverage of the adjusted backfat of the test group; and

M=predicted percent lean calculated for the individual minus the averagepredicted percent lean of the test group.

The number of pigs born alive should be adjusted to a mature sowequivalent by adding the following numbers to the record based on theparity of the female:

TABLE 4 Parity adjustment factors for number born alive. Parity Numberborn alive (L) 1 1.2 2 0.9 3 0.2 4-5 0.0 6 0.2 7 0.5 8 0.9 9 1.1

L=the adjusted number born alive record on the dam minus the average ofthe adjusted number born alive records of her contemporary group;

An individual breeder may wean at any time, but litter weight should berecorded before weaning and as near to 21 days of age as possible forthe most accurate assessment of sow milking ability. Pigs should beweighed between 14 and 28 days and adjusted to a 21-day basis.Post-farrowing litter weights may be adjusted to a 21-day basis by usingthe following multiplicative factors:

TABLE 5 Factors for adjusting litter weight to a 21-day basis. AgeWeighed Factor 10 1.50 11 1.46 12 1.40 13 1.35 14 1.30 15 1.25 16 1.2017 1.15 18 1.11 19 1.07 20 1.03 21 1.00 22 0.97 23 0.94 24 0.91 25 0.8826 0.86 27 0.84 28 0.82

The following equation was used to compute the tabular values and can beused to directly adjust litter weight to a 21-day basis:Adjusted 21-day litter weight=wt.*[2.218−0.0811(age)+0.0011(age²)].

Pigs should be weaned no earlier than 10 days of age; using factors inTable 5 will permit sows to be ranked on the Sow Productivity Index. Thecloser to 21 days of age pigs are weaned, the more accurate theadjustment will be.

If possible, litters should be standardized to between 8 and 12 pigs perlitter within 24, but not later than 48, hours after birth. Pigsselected for transfer should be average in size. Males, rather thanfemales, should be transferred if possible. Standardization of thepost-farrowing weight record will prevent discrimination against a goodmilking sow or gilt that has a lesser opportunity because of smallerthan optimum litter size. The litter weight (already adjusted to a21-day basis) should be standardized to 10 pigs by adding theappropriate value from the following table:

TABLE 6 Factors for adjusting 21-day litter weight for number of pigsafter transfer (number allowed to nurse). Number of pig Adjustmentfactor for safter transfer 21-day litter weight (W) 1-2 104 3 76 4 61 551 6 41 7 30 8 21 9 17 >=10     0

Post-farrowing litter weights should also be adjusted to a mature sowequivalent by adding the following numbers to the record based on theparity of the female:

TABLE 7 Parity adjustment factors for 21-day litter weight. Adjustmentfactor for Parity 21-day litter weight (W) 1 6.2 2 0.0 3 1.0 4 3.8 5 6.26 9.5 7 11.6 8 15.2 >=9     21.5

The adjustment factors in Tables 4-7 were derived for general purposeuse from large data sets. Whenever possible, specific adjustment factorsshould be derived from data sets for specific populations, for use inthose populations specifically. Growth rates must be measured on allintact males and/or all gilts by one of two procedures.

W=the adjusted 21-day litter weight record on the dam minus the averageof the adjusted 21-day litter weight records of her contemporary group;

-   -   (a) Age at a constant weight. If pigs are not weighed on test        but only a final weight is taken, weights should be taken at or        near 250 pounds or some other comparable constant weight. The        equation for adjusting days to a constant weight is:        Adjusted days=actual age+[(desired wt.−actual wt.)*((actual        age−a)/actual wt.)]        -   (where a=50 for boars and barrows, and 40 for gilts).    -   (b) On-test gain. Pigs should be weighed on test at an average        pig weight consistent with the management program of the        operation. Average pig weights of approximately 70 pounds are        recommended. Ranges in starting weights among individual pigs        should be minimized. Off-test pig weight should average at least        160 pounds more than starting weight. If pigs being tested have        undergone segregated early weaning, the test may be started at        an average starting weight of 40 pounds and off-test pig weights        should average at least 190 pounds more than starting weight.

D=adjusted days to 250 pounds measured on the individual minus theaverage of the adjusted days to 250 pounds of the test group;

-   -   (c) Backfat. All pigs should be measured for backfat thickness        at the tenth rib location when they are weighed off-test at 250        pounds. The average of two measurements, taken 2 inches off the        midline on both sides of the pig, should be obtained if a metal        probe or A-mode ultrasound machine is used. If a B-mode        (real-time) ultrasound machine is used, a single measurement is        sufficient. Backfat depth should be measured at the midpoint of        the loin, and should include the skin and all fat layers. If any        other backfat measurements are taken, an explanation should be        given. All measurements should be adjusted to a constant basis        using the formula below:        Adjusted backfat=actual backfat+[(desired wt−actual wt)*(actual        backfat/(actual wt−b))]    -   (where b=−20 for boars, +30 for barrows, and +5 for gilts).

B=backfat measured on the individual, adjusted to 250 pounds, minus theaverage of the adjusted backfat of the test group;

-   -   (d) Loin-Muscle Area. The loin-muscle area (LMA) should be        measured on pigs when they are weighed off-test, within a 30        pound range of the desired weight endpoint (e.g., 250 pounds).        Loin-muscle area should be measured over the 10th rib at a        location 2 inches off the midline. The equation for adjusting        LMA to a constant weight basis is:        Adjusted LMA=actual LMA+[(desired wt−actual wt)*(actual        LMA/(actual wt+155))].    -   (e) Predicted Percent Lean. This trait can be used in place of        backfat in a selection index. Use the following equation to        calculate Predicted Percent Lean (PPL) if pigs are weighed        off-test at 250 pounds:        Adjusted PPL=[80.95−(16.44*adj bf)+(4.693*adj LMA)]*0.54.

M=predicted percent lean calculated for the individual minus the averagepredicted percent lean of the test group.

Given the above adjustment factors, the aforementioned selection indexescan then be calculated as follows:SPI=100+6.5(L)+WEWSPI=100+10(L)MI=100+6(L)+0.4(W)−1.6(D)−81(B)TI−A=100−1.7(D)−168(B)TI−B=100−1.4(D)−106(B)TI−M=100−1.4(D)+12(M)

A. Selection Indexes—Using Expected Progeny Differences

The actual genetic merit of an animal is its breeding value, which isthe sum effect of all its genes. How the breeding value is expressed byeach pig's individual phenotype is dependent on the environmentalconditions under which it is raised. The concept of breeding valuerelates to selection through the fact that genes occur in pairs.Selected animals transmit a sample one-half of their genes (one of eachpair), or one-half of their breeding value, to each offspring. For thisreason, the expected difference between the progeny of an individual andthe original population is one-half the breeding value of thatindividual. Many genetic programs express the genetic merit estimate asexpected progeny differences (EPD), which is one-half the animal's EBV.Therefore, EPD=½ EBV.

EPDs can be based on direct measures of animal performance, and/or onmeasures of performance of relatives to the animal in question(ancestors, siblings, progeny). To calculate EPDs, all availableinformation can be combined in a BLUP statistical procedure. The EPD isa prediction of how progeny of an individual are expected to performrelative to the group or population average (disregarding the otherparent). The EPD for progeny resulting from the mating of a specificmale to a specific female is the sum of the EPDs of the two parents.

EPDs and EBVs are estimates. The more sources of information that can beused in the estimates, the more accurate these estimates will be. Thereliability to be placed on the EPD is associated with the accuracy ofeach EPD. Accuracy is defined as the correlation between the EBV of anindividual and its “true” breeding value. An accuracy value close to 1.0indicates higher reliability for the EPD. The accuracy value reported inmost genetic evaluation programs is a function of the possible change orvariation for that particular trait. A producer may wish to limit theuse of an animal with low accuracy, whereas a boar with many progeny andhence a higher accuracy may be used more extensively. Accuracy valuesare most effective as a tool for risk management because regardless ofaccuracy, EPDs are the best estimates of genetic value currently.

A positive EPD is desirable when selecting for traits such as number ofpigs born alive or 21 day litter weights because a large positive numbertranslates into more pigs per litter or more pounds per litter. Whenselecting against backfat or days to market weight, however, a negativeEPD is desirable because one wishes to reduce the time required to reachmarket weight and the amount of backfat.

If EPDs are reported, animals may be evaluated with selection indexessimilar to those listed above. The simplest index consists of all theEPDs added together. For example, if a producer is interested in littersize, growth and backfat, the index would be:I=100+EPD_(L)+EPD_(D)+EPD_(B)

Use of economic values for each trait can weight the genetic informationfor the relative economic importance of each trait. Using the economicvalues of $13.50 for L, $0.12 for D, and $15.00 for B, for example(economic values given in dollars per 1 unit of change in the trait),would yield the following selection index:I=(100+13.5)*(EPD_(L)−0.12)*(EPD_(D)−15)*EPD_(B)

B. Examples of Calculating Selection Indexes

The following are examples of calculating selection indexes are providedby way of example only.

Calculating a Maternal Index. The gilt to be indexed was born in a firstparity litter of 11 and reared in a litter of 10 which weighed 178 lb at23 days of age. Her weight at 160 days of age was 240 lb, and her B-modeultrasound backfat reading at that weight was 0.9 inches. To calculatethe index value for this gilt, her records must be adjusted to standardconditions. This gilt was born in a first-parity litter, so her dam'srecord for number born alive must be increased by 1.2 pigs (Table 4):11+1.2=12.2 pigs. Since the litter was weighed at 23 days of age, theweight should be multiplied by 0.94 (Table 5). Litter weight should alsobe adjusted for parity by adding 6.2 lb (Table 7). No adjustment forlitter size is necessary because the litter had 10 or more pigs. Thefinal adjusted litter weight is (178*0.94)+6.2=173.5 lb.

The gilt's record for days to 250 lb is calculated using the equation asprovided above. The desired weight is 250 lb, actual weight was 240 lb,actual age was 160 days, and the correction factor for gender is 40days, giving an adjusted days to 250 lb of 165 days. Adjusted backfat iscalculated in a similar manner using the above equation. The adjustedbackfat for this gilt is 0.94 inches.

The adjusted values for the traits must also be calculated for all giltsin the contemporary group and averaged to yield L, W, D, and B. If theaverage adjusted values for this gilt's contemporary group were L=9.4pigs, W=158 lb, D=163 days, and B=1.04 inches, her maternal index is:I=100+6*(12.2−9.4)+0.4(173.5−158)−1.6(165−163)−81(0.94−1.04)=128 indexpoints

Calculating a Terminal Index. Assume one wishes to determine the indexof a boar that required 150 days to reach 250 lb in a contemporary groupthat averaged 165 days. His adjusted backfat (B-mode) was 0.6 inches andthe group average was 0.75 inches. He was born and reared in a secondparity litter of 9 which weighed 200 lb at 22 days. The contemporarygroup adjusted values for litter size and weight are 9.1 and 185,respectively. To calculate the terminal index, one uses the adjustedvalues already determined in the selection index for B-mode backfatscans as provided above:I=100−1.4(150−165)−106(0.6−0.75)=137 index points

Note that one can ignore the litter information because this boar willbe used to sire only market pigs. If one were interested in using him tosire replacement gilts, one would need to use the maternal index.

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments using sex-selectedsperm cells to increase the genetic progress of a line, breed or herd,including, but not limited to, the best mode of the invention.

As such, the particular embodiments or elements of the inventiondisclosed by the description or shown in the figures or tablesaccompanying this application are not intended to be limiting, butrather exemplary of the numerous and varied embodiments genericallyencompassed by the invention or equivalents encompassed with respect toany particular element thereof. In addition, the specific description ofa single embodiment or element of the invention may not explicitlydescribe all embodiments or elements possible; many alternatives areimplicitly disclosed by the description and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of “sorter” should beunderstood to encompass disclosure of the act of “sorting”—whetherexplicitly discussed or not—and, conversely, were there effectivelydisclosure of the act of “sorting”, such a disclosure should beunderstood to encompass disclosure of a “sorter” and even a “means forsorting.” Such alternative terms for each element or step are to beunderstood to be explicitly included in the description.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity. As such, the terms “a”or “an”, “one or more” and “at least one” can be used interchangeablyherein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes for example the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentapplication or continuation, division, or continuation in partapplication thereof, or to obtain any benefit of, reduction in feespursuant to, or to comply with the patent laws, rules, or regulations ofany country or treaty, and such content incorporated by reference shallsurvive during the entire pendency of this application including anysubsequent continuation, division, or continuation in part applicationthereof or any reissue or extension thereon.

What is claimed is:
 1. A method of increasing the genetic progress of aline or breed of swine within a genetic nucleus comprising the steps of:providing a boar from a genetic nucleus and a sow from a geneticnucleus, wherein said boar and said sow are from a line or breed withinsaid genetic nucleus and wherein said line or breed of said boar andsaid sow are the same line or breed; collecting a semen sample from saidboar; sorting said semen sample into at least two subpopulations ofsperm cells, wherein at least 80% of a first subpopulation bearsX-chromosomes or Y-chromosomes; inseminating said sow with sperm cellsfrom said first subpopulation to produce offspring from said sow;genotyping said offspring to identify one or more genetic markers;selecting one or more of said offspring as a parent in the line or breedin the genetic nucleus based on the one or more identified geneticmarkers.
 2. The method of claim 1 wherein said line or breed is a giltline and said first subpopulation bears X-chromosomes.
 3. The method ofclaim 1 wherein said line or breed is a boar line and said firstsubpopulation bears Y-chromosomes.
 4. The method of claim 1, wherein theone or more genetic markers are single nucleotide polymorphisms.
 5. Themethod of claim 4, wherein the single nucleotide polymorphisms arecorrelated with feed efficiency, average daily gain or carcass lean.